Method for making cheese

ABSTRACT

The present invention relates to a method for making Cheddar type and Continental type cheese with an adjunct culture comprising a  Lactobacillus helveticus  strain.

FIELD OF INVENTION

The present invention relates to a method for making cheese, especiallyCheddar type and continental type cheese, with a Lactobacillushelveticus culture as adjunct culture.

BACKGROUND OF INVENTION

Cheddar type cheeses are made with either mesophilic cultures or amixture of mesophilic and thermophilic cultures. They are dry saltedafter the milling step. An example of a rather traditional Cheddar makeis given below to illustrate the manufacturing process (according toWalstra et al, 2006 Dairy Science and Technology/Second edition, Taylor& Francis, page 713): Milk is pasteurized (15 sec/71° C.), filled intothe cheese vat with a temperature of 30° C. and pre-acidified withaddition of starter culture for 40 min at 30° C. Rennet is added andcoagulation process takes 35 min at 30° C. The coagulum is cut (10 min)and during 30 min of stirring the curd/whey is heated up to scaldingtemperature of 40° C. The scalding temperature is held for 60 furtherminutes. Then the curd settles, fuses into a compact mass and whey istaken off (30 min). Afterwards, the cheddaring takes place (100 min)where the curd mass is cut into large strips that are piled up andturned. Prior to salting, the curd is milled into small strips. Salt isadded and mixed with the curd strips. The salted curd is then filledinto moulds and pressed (16 h/2 bars). After a certain drying phase,cheeses are packed and ripened at relatively low temperature such as 8to 11° C.

Traditionally, cheeses of the continental type are made with mesophilestarter cultures. An example of a cheese of the continental type is theSamsø cheese. A Samsø cheese (30% fat in dry matter) is made accordingto the following steps: Milk is standardized in fat content (e.g. 1.6%fat), pasteurized (20 sec/72° C.) and filled into the cheese vat with atemperature of 31° C. KNO₃ and CaCl₂ may be added to the milk. Milk isafterwards inoculated with the mesophile starter culture. Rennet isadded and coagulation process takes 45 min at 31° C. The coagulum is cut(5 min) and 35% of the whey is drained off. Hot water is added and thescalding is made at 37° C. The stir out phase takes 40 min andafterwards the whey is drained off and the curd is transferred intomoulds. The curd is pressed in the moulds at 4 bar, and then at 6 barfor 70 min in total. After a subsequent resting phase cheese temperaturefalls to 12° C. Subsequently cheeses are salted in a brine (20 hours).

In the manufacturing of fermented milk product there is a constant needfor alternative and improved manufacturing method. Such desired methodstypically aim at improving the manufacturing process e.g. by reducingcost, increasing speed of the overall process and/or improvecharacteristics of the final product. Desired product improvementsinclude all known product quality parameters such as taste, texture,flavour etc. In fermented products the manufacturing process includesthe addition of a starter culture performing the specific fermentation.In cheese production, the objective of the starter culture is toprimarily acidify the raw material, milk. Often, it is not possible tooptimize the performance of the primary starter in a way that securesoptimal taste/flavour of the final product.

In the case of cheese production, often a ripening step is included inthe production process. During the ripening phase proteolysis, aminoacid catabolism and lipolysis are key factors for texture and flavourdevelopment in the cheese product. The ripening phase should result inthe development of the desired organoleptic properties of the cheese ina short time frame. As a result it has been suggested to apply “adjunctcultures” in the manufacturing of fermented milk products aiming atimproving the characteristics of the product such as texture and/orflavour. This is of special relevance in reduced-fat or low-fat cheeses.Adjunct cultures are expected to improve the characteristics of theproduct such as texture and/or flavour but not to interfere with theacidification of milk caused by the primary starter culture. Adjunctcultures influence the cheese ripening process trough their enzymaticsystems involving among others proteinases, peptidases, aminopeptidases,aminotransferases, esterases and lipases. The enzymatic potential isspecies and/or strain dependent.

-   -   Adjunct cultures are often Lactobacillus strains which are shown        to contribute to the flavor development. The use of        Lactobacillus helveticus as an adjunct culture to cheese has        been studied. Nevertheless Lactobacillus helveticus as an        adjunct culture can only be used at limited inoculation rates        due to its impact on the acidification profile. This is        especially relevant in cheese technologies where the scalding        temperature is high enough to allow significant growth of the        thermophile Lactobacillus helveticus adjunct culture (as e.g.        40° C. in a Cheddar cheese make). Different approaches were        proposed to overcome this problem. One approach is to use        attenuated (non viable) adjunct cultures. The most investigated        methods for attenuation are of physical character as e.g. heat        shock and freeze shock. The drawback of this approach is that        the attenuation process is an extra step during culture        preparation that has to be carried out either at the culture        producer or the cheese producer under reproducible conditions.        It should be emphasized that changes in the acidification        process during a cheese make will without any further adaptation        of the cheese making process impact on whey drainage from the        cheese curd, the content of minerals in the curd/cheese and the        minimum pH during the cheese make. This has important unwanted        consequences on the cheese composition, cheese ripening and the        obtained characteristics of the product such as texture and/or        flavour.

SUMMARY OF INVENTION

It has surprisingly turned out that it is possible to obtain aLactobacillus adjunct culture which does not influence milkacidification during cheese making. This Lactobacillus culture is amutant of an acidifying strain, and has turned out to be non-acidifying.According hereto, the present inventors have brought forward a method toimprove the texture and/or taste and/or flavour of cheeses, especiallyof the cheddar type and the continental type which method implies usinga non-acidifying thermophilic Lactobacillus helveticus strain as adjunctculture without influencing milk acidification. The non-acidifyingLactobacillus helveticus strains were obtained by mutation of anacidifying mother strain DSM 19500.

During cheese production, the non-acidifying mutants may be added to thecheese milk together with the primary starter cultures.

It was surprisingly observed that the non-acidifying Lactobacillushelveticus mutants maintained the mother strain's ability to improvecheese texture and/or taste and/or flavour, especially to debittercheese and to introduce the typical Lactobacillus helveticus “sweet”flavour note in ripened cheese. The mutant was used for the cheese makeas a non attenuated adjunct culture, so no extra attenuation process wasneeded.

In accordance with the surprising finding, the present invention relatesto a process for producing cheese (eg full fat, reduced fat and low fatcheese), which comprises:

adding to milk

-   -   a starter culture, such as culture comprising a strain belonging        to a genus selected from the group consisting of: Lactococcus,        Leuconostoc, Pediococcus, Streptococcus, and Enterococcus, and    -   a non-acidifying Lactobacillus helveticus strain as adjunct        strain;    -   a coagulant, such as a milk-clotting enzyme;        heating the mixture to a temperature (or maintaining the        temperature) in the range of 30 to 45 degrees C.

DETAILED DESCRIPTION OF INVENTION

In a first aspect, the present invention relates to a process forproducing cheese, which comprises:

adding to milk

-   -   a starter culture, such as culture comprising an acidifying        strain belonging to a genus selected from the group consisting        of: Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and        Enterococcus; and    -   an adjunct culture comprising a non-acidifying Lactobacillus        helveticus strain; and        heating the mixture to a temperature (or maintaining the        temperature) in the range of 30 to 45 degrees C., such as in the        range 35 to 43 degrees C. or in the range 37-43 degrees C.

An interesting embodiment relates to a process for producing cheese(including reduced and low fat cheese), which comprises:

adding to milk

-   -   a starter culture, such as culture comprising an acidifying        strain belonging to a genus selected from the group consisting        of: Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and        Enterococcus;    -   an adjunct culture comprising a non-acidifying Lactobacillus        helveticus strain; and    -   a coagulant, such as a milk-clotting enzyme; and        heating the mixture to a temperature (or maintaining the        temperature) in the range of 30 to 45 degrees C., such as in the        range 35 to 43 degrees C. or in the range 37-43 degrees C.

An other interesting embodiment relates to a process for producingcheese (including reduced and low fat cheese), which comprises:

adding to milk

-   -   a starter culture, such as culture comprising an acidifying        strain belonging to a genus selected from the group consisting        of: Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and        Enterococcus;    -   an adjunct culture comprising a non-acidifying Lactobacillus        helveticus strain; and    -   a coagulant, such as a milk-clotting enzyme; and        heating the mixture to a temperature (or maintaining the        temperature) in the range of 30 to 45 degrees C., such as in the        range 35 to 43 degrees C. or in the range 37-43 degrees C. and        holding the mixture at that temperature range for 5 to 70        minutes immediately before whey removal or pre-pressing under        whey.

Also, the invention relates to a process for improving the textureand/or taste and/or flavour of cheese, and a process for improvingcheese quality, the processes comprising adding to milk

-   -   a lactic acid bacteria culture comprising a strain belonging to        a genus selected from the group consisting of: Lactococcus,        Leuconostoc, Pediococcus, Streptococcus, and Enterococcus, and    -   a non-acidifying Lactobacillus helveticus strain; and;    -   a coagulant, such as a milk-clotting enzyme; and        heating the mixture to a temperature (or maintaining the        temperature) in the range of 30 to 45 degrees C., such as in the        range 35 to 43 degrees C. or in the range 37-43 degrees C.

In a preferred embodiment, the non-acidifying Lactobacillus helveticusstrain is not able to lower the pH more than 1.5 pH Units (such as morethan 1.3) from start pH 6.5 after 10 hours incubation at 37° C. wheninoculated from a fresh over night culture at inoculation dose 1.5×10⁷cfu/ml into 200 ml milk, especially into milk prepared from 9.5% skimmilk powder rehydrated in water (heat treated at 140° C./8 sec and 100°C./30 min). Thus, in the most preferred embodiment, the non-acidifyingLactobacillus helveticus strain is not able to lower the pH more than1.3 pH Units from start pH 6.5 after 10 hours incubation at 37° C. wheninoculated from a fresh over night culture at inoculation dose 1.5×10⁷cfu/ml into 200 ml milk prepared from 9.5% skim milk powder rehydratedin water (heat treated at 140° C./8 sec and 100° C./30 min).

The non-acidifying Lactobacillus helveticus strain may be a mutant of anacidifying strain (which in the present context is a strain able tolower the pH more than 1.3 (or more than 1.5) pH Units from start pH 6.5after 10 hours incubation at 37° C. when inoculated from a fresh overnight culture at inoculation dose 1.5×10⁷ cfu/ml into 200 ml milkprepared from 9.5% skim milk powder rehydrated in water (heat treated at140° C./8 sec and 100° C./30 min). In a embodiment, the non-acidifyingLactobacillus helveticus strain is a mutant of an acidifyingLactobacillus helveticus strain, preferably a mutant having an at leastas high cell wall bound protease activity as the mother strain(determined by identical method as disclosed in example 1c). In apresently preferred embodiment, the non-acidifying Lactobacillushelveticus strain is a mutant of the acidifying strain DSM 19500, e.g.the mutant DSM19501.

In an important embodiment of the process of the invention, the starterculture is added in an amount of at least 10³ CFU per ml milk, and/orthe Lactobacillus helveticus strain is added in amount of at least 10⁴(such as at least 10⁵, 10⁶, at least 10⁷ or 10⁸) CFU per ml milk.

In the process of the invention the starter culture may comprisebacteria belonging to a strain selected from the group consisting of:Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus pentosaceus,Lactobacillus casei, Lactobacillus paracasei, Streptococcusthermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus delbrueckii subsp. lactis and Lactobacillusacidophilus, preferably the strain is Lactococcus lactis subsp. lactis,Lactococcus lactis subsp. cremoris, or Lactococcus lactis subsp. lactisbiovar. diacetylactis.

The cheese be made by the process may be a cheddar type cheese, or acontinental type cheese (eg Gouda, Danbo, Havarti etc), includinglow-fat cheese, and thus it is presently preferred that no bacteriabelonging to the genus Propionibacterium are added to the milk,especially in a concentration over 10² cfu per ml, but the processinstead comprises the further step of pressing the mixture obtained inthe heating step, either before or after salting. The pressed and saltedcheese is preferably kept at a temperature in the range of 1 to 25degrees C., or the pressed and salted cheese is not maintained at atemperature over 25 degrees C. for more than 2 hours.

In an other aspect, the present invention relates to a cheese (includinga low-fat cheese) obtainable by the process of any preceding claim, suchas a cheddar type cheese.

In the last aspect, the present invention relates to strains that can beused in the process of the invention, and strains that can be used asstarting material for a non-acidifying mutant. Thus, the presentinvention relates to an acidifying Lactobacillus helveticus strainselected from the group of DSM19500, DSM 18879, DSM 18880, DSM 18881,DSM 18871, DSM 18872, DSM 18873, DSM 18883, DSM 18884 and the mutantsand variants of any of these, esp. non-acidifying mutants and variants,and to the Lactobacillus helveticus strain DSM1 9501 and mutants orvariants thereof, such as non-acidifying mutants and variants.

DEFINITIONS

By the term “milk” is understood a composition comprising lactealsecretion obtained from any mammal, such as an animal of a speciesbelonging to the subfamily Bovinae (which includes the domestic cow (Bostaurus) and buffalo); an animal of a species belonging to the subfamilyCaprinae (which includes goat and sheep); or an animal of the speciesCamelidae (which includes camels). Optionally the milk is acidified,e.g. by addition of an acid (such as citric, acetic or lactic acid) orby addition of an acid producing microorganism. The milk may be raw orprocessed, e.g. by filtering, sterilizing, pasteurizing, homogenizing,fractionating (e.g. reducing the fat content of the milk), etc, or itmay be reconstituted dried milk. An important example of “milk”according to the present invention is pasteurized cow's milk. It isunderstood that the milk may be acidified, mixed or processed before,during and/or after the adding of bacterial cultures.

The term “coagulant” refers to any kind of milk clotting agent, such asa native enzyme derived from microbial, vegetable or animal tissuesources or a milk clotting enzyme provided as a gene product ofrecombinant cells expressing a milk clotting enzyme of animal ormicrobial origin. The term includes bovine chymosin purified fromabomasum tissue or made by fermentation (e.g. CHY-MAX® or CHY-MAX® M).

The term “cheese” refers to a product prepared by contacting optionallyacidified milk (e.g. by means of a lactic acid bacterial culture) with acoagulant, and draining the resultant curd. Cheeses and theirpreparation are described in “Cheese and Fermented Milk Foods”, by FrankV. Kosikowski.

The term “cheese of the cheddar type” should be understood as cheeses ofthe types such as Cheddar, Territorials, American Cheddar, Monterey Jackand Colby, and/or cheeses made by a process which includes heating thecurd to a temperature that does not exceed 45 degrees C. In the presentcontext, cheese of the cheddar type is characterized by:

-   -   Fat in Dry matter: 10-60%    -   Humidity: 34-42%    -   Salt content: 1.5-2.5%    -   Cheddaring and subsequent Milling step    -   Salting after milling but before pressing    -   Pressing step

The term “cheese of the continental type” should be understood ascheeses of the types, such as Gouda, Danbo, Edam, St. Paulin, Raclette,Fontal etc and/or cheeses made by a process which includes heating thecurd to a temperature that does not exceed 45 degrees C. In the presentcontext, cheese of the continental type is characterized by:

-   -   Fat in Dry matter: 10-60%    -   Water content: 35-57%    -   Water in Fat free cheese matter: 53-63%    -   Salt content: 1-3.5%    -   Pressing step during cheese manufacture process    -   Salting after pressing most often in a brine

The term “reduced fat cheese” refers to cheese having a fat contentreduced to 32% fat in dry matter or less, down to 25% fat in dry matter,and the term “low fat cheese” refers to cheese having a fat contentreduced to 25% fat in dry matter or less. The person skilled in the artis familiar with the adjustment of the milk fat content in respect tovarying protein content of the milk. Fat content in cheese can bedetermined after van Gulik method ISO 3433, commonly known by theskilled person of the art. Example for Gouda cheese:

full fat cheese: 45% fat in dry matter/ca. 3.1% fat in milkreduced fat cheese: 30% fat in dry matter/ca. 1.6% fat in milklow fat cheese: 15% fat in dry matter/ca. 0.7% fat in milk

As used herein the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid. The industrially most useful lactic acidbacteria are found among Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pediococcus spp. Additionally,lactic acid producing bacteria belonging to the group of the strictanaerobic bacteria, Bifidobacterium spp., which are frequently used asfood cultures alone or in combination with other lactic acid bacteria,are generally included in the group of lactic acid bacteria. The term“CFU” (or “cfu”) is short for cell forming units.

The term “starter culture” relates to any bacterial culture that issuitable for use in milk acidification, especially lactic acid bacteriasuch as Bifidobacteria, Lactobacilli, Lactococci, Leuconostocs,Micrococci and Pediococci. It will be appreciated that the term starterculture may encompass a culture containing a single strain of bacterium,or more than one bacterial strain. The term may also include geneticallymodified organisms (GMO's). In any event, the term is well known in theart and the invention extends equally to all known starter cultures. Theterm includes bacterial cultures containing a strain of a genus selectedfrom the group consisting of Lactococcus, Lactobacillus, Micrococcus,Leuconostoc, Pediococcus, Streptococcus, Enterococcus, etc. such as astrain of a species selected from the group consisting of: Lactococcuslactis (incl. Lactococcus lactis subsp. lactis, Lactococcus lactissubsp. cremoris, and Lactococcus lactis subsp. lactis biovar.diacetylactis), Leuconostoc mesenteroides (incl subsp. cremoris),Pediococcus pentosaceus, Lactobacillus casei (incl. subsp. casei) andLactobacillus paracasei (incl. subsp. paracasei), Streptococcusthermophilus, Enterococcus faecium, Lactobacillus helveticus,Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckiisubsp. lactis and Lactobacillus acidophilus. Other useful bacterialspecies are Bifidobacterium species including B. bifidum, B. lactis andB. longum, Streptococcus faecium, Leuconostoc lactis.

As previous mentioned, lactic acid bacteria are essential in the makingof nearly all fermented milk products e.g. cheese, and they are normallysupplied to the dairy industry either as frozen or freeze-dried culturesfor bulk starter propagation or as so-called “Direct Vat Set” (DVS)cultures, intended for direct inoculation into a fermentation vessel orvat for the production of a dairy product. Such cultures are in generalreferred to as “starter cultures” or “starters”.

Commonly used starter culture strains of lactic acid bacteria aregenerally divided into mesophilic organisms having optimum growthtemperatures at about 30 to 35° C. and thermophilic organisms havingoptimum growth temperatures in the range of about 40 to about 45° C.Typical organisms belonging to the mesophilic group include Lactococcuslactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostocmesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcuslactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp.casei and Lactobacillus paracasei subsp. paracasei. Thermophilic lacticacid bacterial species include as examples Streptococcus thermophilus,Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis,Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus,Lactobacillus delbrueckii subsp. lactis and Lactobacillus acidophilus.

Additionally, species of Propionibacterium are used as dairy startercultures, in particular in the manufacture of cheese.

“Adjunct cultures” are in this context defined as strains of cheeserelated microorganisms that are added to the cheese milk to improve thesensory quality of cheese in terms of texture and/or taste and/orflavour. Adjuncts cultures are specifically selected for their abilitiesto improve the sensory quality of cheese and are intentionally added tothe cheese milk by the cheese maker at preferably 10² to 10⁸ cfu/mlmilk. Adjunct cultures influence the cheese ripening process troughtheir enzymatic systems involving among others proteinases, peptidases,aminopeptidases, aminotransferases, esterases and lipases. The enzymaticpotential is species and/or strain dependent.

Adjunct cultures have become an important tool for the cheesemanufacturer in achieving cheeses with improved taste and/or textureand/or flavour. The need to improve flavour development especially inreduced or low-fat cheese has created an increased interest in theutilization of adjunct cultures.

The term “non-acidifying” in the present context refers to a strain thatdoes not lower the pH of (bovine) milk more than 1.5, or presentlypreferred, not more than 1.3 pH Units from start pH 6.5 after 10 hoursincubation at 37° C., preferably under the following conditions:

-   -   inoculated from a fresh over night culture at inoculation dose        1.5×10⁷ cfu/ml    -   into (e.g. 200 ml) milk prepared from 9.5% skim milk, e.g powder        rehydrated in water, and preferably heat treated at 140° C./8        sec and 100° C./30 min. The person skilled in the art should        understood how to incubate the L. helveticus strain and assess        the strain's acifying properties, and he will find guidance in        the assay: assessment of acidification activity of mutants by        direct pH measurement in milk, see example 2.

In the present context, the term “mutant” should be understood as astrain derived from a strain of the invention by means of e.g. geneticengineering, radiation and/or chemical treatment. It is preferred thatthe mutant is a functionally equivalent mutant, e.g. a mutant that hassubstantially the same, or improved, non-acidifying properties as themother strain. Such a mutant is a part of the present invention.Especially, the term “mutant” refers to a strain obtained by subjectinga strain of the invention to any conventionally used mutagenizationtreatment including treatment with a chemical mutagen such as ethanemethane sulphonate (EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UVlight or to a spontaneously occurring mutant.

In the present context, the term “variant” should be understood asstrain which is functionally equivalent to a strain of the invention,e.g. having substantially the same, or improved, non-acidifyingproperties. Such variants, which may be identified using appropriatescreening techniques, are a part of the present invention.

The use of the terms “a” and an and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “having”, “including” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

DRAWING

FIG. 1 depicts the cell wall bound proteinase activity of wild type andone mutant of Lb. helveticus DSM19500. The cell wall-associatedproteinase activity is shown as specific activity (Fluorescence units/ODunit), cf. the example 1.

FIG. 2 depicts the acidification profile in milk of the mother stainLactobacillus helveticus DSM19500 and Lactobacillus helveticus mutantstrain DSM19501, cf. the example 2.

FIG. 3 depicts the acidification profile during cheese make of controlcheese, cheese made with the mother stain Lactobacillus helveticusDSM19500 as adjunct and cheese made with Lactobacillus helveticus mutantstrain DSM19501 as adjunct, cf. the example 3.

FIG. 4 depicts the flavour profile of control cheese, cheese made withLactobacillus helveticus mother strain DSM19500 and cheese made withLactobacillus helveticus mutant strain DSM19501, cf. the example 3.

DEPOSITS AND EXPERT SOLUTION

The Lactobacillus helveticus mother strain was deposited Jul. 4, 2007 atDeutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,Inhoffenstr. 7B, D-38124 Braunschweig (DSM) and given the accessionnumber DSM 19500. The Lactobacillus helveticus mutant strain wasdeposited Jul. 4, 2007 at DSM and given the accession number DSM19501.The deposits were made according to the Budapest treaty on theinternational recognition of the deposit of microorganisms for thepurposes of patent procedure.

The applicant requests that a sample of the deposited microorganismsstated above may only be made available to an expert, until the date onwhich the patent is granted.

REFERENCES

-   Walstra et al, 2006 Dairy Science and Technology/Second edition,    Taylor & Francis Cheese and Fermented Milk Foods, by Frank V.    Kosikowski.-   El-Soda et al, 2000. Adjunct cultures: Recent Developments and    Potential Significance to the Cheese Industry. Journal of Dairy    Science. 83: 609-619.

All references cited in this patent document are hereby incorporatedherein in their entirety by reference.

EXPERIMENTAL Conventional Cheese Manufacturing

Most cheese productions include a pasteurization and fat standardizationof cheese milk prior to the inoculation of lactic acid bacteria starterculture (either as liquid bulk or as concentrated bacteria (frozen orfreeze-dried), e.g. Chr. Hansen's Direct Vat Set). Starter cultureinoculation takes typically place at temperatures from 30 to 35° C. Theso-called pre-ripening time may vary between cheese types, but istypically in the range of 10 to 60 min before a coagulant is added toset the milk having a typically setting time in the range of 15 to 60min.

When the curd is formed it is cut into cubes (typically 5-20 mm) inorder to facilitate syneresis of whey and concentration of milkconstituents during a gentle agitation. Many cheese varieties undergo anincrease of temperatures in the cheese vat (typically 36-45° C.). Thisis normally done with two purposes: 1) to increase the speed ofsyneresis and reduced the final moisture in cheese and 2) to affect thestarter culture either by increasing the acidification speed ofespecially thermophilic LAB and/or by inducing lysis of mesophilic LAB.During the process in the cheese vat, parts of the whey may be removedand water may be added. When the curd grains obtain the right textureand acidity (typically pH 5.0 to 5.5) the grains are moulded (with orwithout whey removed) and pressed into shapes depending on the specificcheese variety. Most cheeses are salted, which can be done either before(typically dry salting) or after (brine-salting) the moulding. Ripeningand storage conditions vary between different cheese varieties.

Sensory Analysis

According to International Standards (ISO 5492:1992 Sensoryanalysis—vocabulary) sensory perception include the attributes “taste”,“flavour”, “odour” and “aroma”. Aroma and odour are primarily associatedwith the perception by the olfactory organ (nose) prior to and duringeating. Taste is defined as the basic tastes perceived by the taste budsin the mouth. Typically, taste is described as “sweet”, “sour/acid”,“salt” and “bitter”. Flavour is defined as a complex combination of theolfactory, gustatory (taste) and trigeminal (feeling) sensationsperceived during eating. When food products are to be sensory evaluatedit is custom to focus on the perception of taste and flavour.

Thus, when the expression “improved/altered taste and/or flavour” isused herein, it is to be understood as the improved/altered taste and/orflavour as perceived and described by the sensory panel evaluating thecheese of the invention. This should not be taken as an exclusion ofpossibly altered odour and/or aroma but merely as a simple means todescribe the fermented milk product of the invention.

For illustration, in working example 3 herein, one suitable sensoryevaluation method is the “Sensory profile”. Preferably, the test isperformed according to the International Standard (ISO 13299:2003Sensory analysis—Methodology—General guidance for establishing a sensoryprofile).

This standard describes a guidance on the steps that are common to allsensory profiling. Sensory profiles can be established for products suchas e.g. foods and beverages. Sensory profiling is based on the conceptthat the sensory impression made by the sample consists of a number ofidentifiable sensory attributes (descriptors), each of which is presentto a larger or smaller degree. The list of relevant sensory descriptors,each with its intensity value, is the sensory profile. Sensory profilingcan be used to compare a product/sample with a standard or with othersimilar products, also across all of the senses. Thus, the method issuitable for the present purpose i.e. evaluate the effect of use of anadditional ingredient i.e. an adjunct culture in the production of afermented milk product.

As will be illustrated in the example 3 herein, the assessors of thesensory panel were able to compare the product made according to thepresent invention using a non-acidifying Lactobacillus helveticus asadjunct culture in comparison to a product made with an acidifyingLactobacillus helveticus and the product made without adjunct culture.

Example 1 Generation of Non-Acidifying Lactobacillus helveticus Mutantsand Verification of their Acidification Behaviour in Milk

a) Generation of Non-Acidifying Mutants from Lactobacillus helveticusDSM19500 by Ethyl Methane Sulphonate (EMS) Mutagenesis

-   -   From frozen stock ampoule, 100 μl of strain DSM19500 were        inoculated into 10 ml M17 broth+1% lactose. The culture was        incubated at 37° C. (anaerobic) overnight.    -   150 μl Ethyl Methane sulphonate were added to the 10 ml        overnight culture and the tube was sealed with para-film. The        EMS-culture was incubated in a closed incubator-box at 37° C.        rotating slowly for 2 hours. 200 μl EMS-culture was inoculated        into 10 ml M17 broth+1% lactose. The culture was incubated at        37° C. (anaerobic) overnight.    -   200 μl 87% glycerol were added to 1000 μl EMS-overnight-culture,        mixed thoroughly and the stocks were stored at minus 80° C. 100        μl sample diluted to 10⁻⁴-10⁻⁷ was spread on petri-dishes with        20 ml M17 agar+1% w/v lactose+0.5 mg/ml streptomycin sulphate        and 100 μl sample diluted to 10⁻⁴-10⁻⁷ was spread on        petri-dishes with 20 ml M17 agar+1% lactose and incubated at 37°        C., anaerobically, over night.    -   The mutagenesis frequency was checked by counting colonies on        the streptomycin plates and comparing with numbers of colonies        on plates without streptomycin. The frequency of streptomycin        resistant colonies should increase if the mutagenesis has worked        satisfactorily. Furthermore, the number of colonies on plates        without streptomycin was used for calculating the CFU/ml in        order to obtain 3000 colonies when plating the mutagenized        culture on Q-trays (Genetix Ltd., UK) for picking by an        automated colony picker.    -   Q-trays contained MRS agar and were incubation at 37° C. over        night anaerobically. Colonies were transferred by the colony        picker to microtiter plates for assessment of acidification        activity in milk.

b) Assay for Assessment of Acidification Activity of Mutants inMicrotiter Plates During Screening Process

Milk used for acidification experiments was prepared from 9.5% skim milkpowder rehydrated in water and subsequently heat treated at 140° C./8sec and 100° C./30 min.

Microtiter plates (384 wells) with milk containing 0.0476 mg/mlbromocresol purple (Na-salt) and 0.0476 mg/ml bromocresol green(Na-salt) was used for screening for mutants unable to grow in milk(method see WO2005/068982). Microtiter plates were incubated at 37° C.over night, anaerobically. If the milk changed color from blue toyellow, the strain was able to grow and made acid from lactose henceacidifying the media. If the color of milk stayed blue the strain wasnot able to produce acid in milk.

Stability of the non-acidifying isolates was tested by re-stricking 3times on indicator-milk agar plates: Agar plates were prepared from milkcontaining 0.0476 mg/ml bromocresol purple (Na-salt) and 0.0476 mg/mlbromocresol green (Na-salt) as described above. 3.375 g bacto agar weredissolved in 25 ml milliQ water and added to 200 ml of hot indicatormilk. The isolates were incubated at 37° C. and it was verified that thenon-acidifying isolates did not start acidifying, but stayed blue incolor (pH indicator) and grew poorly on the indicator milk plates. Themother strain DSM19500 turned yellow on the indicator milk agar plates.

c) Assessment of Proteinase Activity

The selected isolates, unable to grow in milk, were inoculated in a96-well microtiter plate containing MRS media. The microtiter plate wasincubated at 37° C., anaerobically over night. The cell-wall associatedproteinase activity was determined in a liquid handling robot asdescribed below:

Solution A: 100 mM MES buffer, pH 5.5 containing 50 mM CaCl₂ (19.52 gMES and 7.35 g CaCl₂. Add water to 800 ml, adjust pH to 5.5 with 1 MHCl. Fill with water to 1000 ml)

Solution B: 5 mg/ml FITC-labeled casein (Sigma C3777), prepared inMilli-Q water

Solution C: 5% w/v TCA (5 g TCA. Add Milli-Q water to 100 ml)

Solution D: 500 mM Tris-HCl, pH 8.5 (60.55 g Tris-HCl. Add water to 800ml, adjust pH to 8.5 with 1 M HCl. Fill with water to 1000 ml)

Microtiter plates: Nunc (Product no. 167008), Nunc Black (Product no.237105), MJR PCR plate (V-shaped, product no. HSP-9665)

Tecan Liquid Handling Robot with 8- and 96-pipetting units,fluorescence/absorbance reader (Genious or Vic2), Galaxy incubator, tworobotic arms, cooling units and a microtiter-plate centrifuge

-   -   1. The microtiter plate was centrifuged (2200 rpm. for 2        minutes, 6° C.) and 180 μl supernatant was asprited to waste.        The cells were resuspended in 180 μl solution A (precooled to 4°        C.). This washing step was repeated once more.    -   2. 4 μl of FITC-labeled casein (precooled to 4° C.) were        dispensed to a MJR PCR microtiter plate.    -   3. 20 μl of cell suspension were dispensed to the MJR PCR        microtiter plate. The MJR PCR microtiter plate was incubated for        3 hours or 6 hours at 37° C. (one microtiter plate per        incubation time).    -   4. 57.5 μl of solution C were dispensed to the MJR PCR        microtiter plate. The MJR PCR microtiter plate was incubated for        1 hour at room temperature. The MJR PCR microtiter plate (2200        rpm. for 2 minutes, 6° C.) was centrifuged and 45 μl were        dispensed from the MJR PCR microtiter plate to a Nunc Black        microtiter plate. 125 μl of solution D were aspirated to the        Nunc Black microtiter plate and mixed.    -   5. The fluorescence was read (Genious reader) of the Nunc Black        microtiter plate using excitation and emission wavelengths of        485 and 535 nm, respectively.

At the same time the optical density of the washed cell suspensions isdetermined:

-   -   20 μl of three times washed and finally resuspended cells from        step 1. were aspirated to a Nunc microtiter plate. 80 μl of        solution A was added to the Nunc microtiter plate and mixed.        Optical density was measured of the cell suspensions at 595 nm.

FIG. 1 shows the cell wall bound proteinase activity of wild the typestrain Lactobacillus helveticus DSM19500 and the mutant DSM19501. Thecell wall-associated proteinase activity is shown as specific activity(Fluorescence units/OD unit). Per strain, 8 independent growingsubcultures were made and standard deviations are given for these 8replicates per strain.

It was shown that the non-acidifying mutant DSM19501 has comparable cellwall bound proteinase activity than the mother strain.

Example 2 Assessment of Acidification Activity of Mutants by Direct pHMeasurement in Milk

As a start, working ampoules were made by incubating the strains in MRSbroth at 37° C. over night. The pH was adjusted to pH 6.2 with NaOH and20% sterilized Glycerol. The ampoules of each strain were stored frozenat −80° C. The strains were inoculated from the frozen ampoules at 1%v/v into MRS growth medium and incubated at 37° C. over night to obtain1.5×10⁹ cfu/ml.

2 ml of the over night culture were inoculated in 200 ml milk andincubated at 37° C. Milk used for acidification experiments was preparedfrom 9.5% skim milk powder rehydrated in water and subsequently heattreated at 140° C./8 sec and 100° C./30 min. pH was measuredcontinuously with pH probes connected to a datalogger.

The mother strain acidified down to pH 4.2 at 10 hours whereas thenon-acidifying strain stayed at higher pH (see FIG. 2).

Example 3 Use of Non-Acidifying Lactobacillus helveticus Mutant DSM19501as Adjunct Culture in the Production of Cheddar Cheese

Cheddar cheeses (50% fat in dry matter and 54% moisture in non-fatsubstance) were made from pasteurized (72° C. for 15 s) bovine milkusing chymosin (CHY-MAX™ Plus, Chr. Hansen A/S) and a frozenCheddar-starter culture F-DVS RST-630 composed of Lactococcus lactissupsp. lactis and Streptococcus thermophilus (0.008% w/w F-DVS RST630,Chr. Hansen A/S).

Experimental cheeses were made with the starter culture alone, incombination with Lactobacillus helveticus mother strain DSM19500 or incombination with the non-acidifying Lactobacillus helveticus mutantstrain DSM19501 respectively. The inoculation level of the Lactobacillushelveticus strains was 6×10⁶ cfu/ml milk.

The manufacturing method used was as previously described (conventionalcheese manufacture) with a starter inoculation at 32° C., a pre-ripeningtime of 45 minutes and a setting time of 45 minutes. The coagulum wascut in cubes (5×5×5 mm), stirred for 15 min and then temperatureincreased to 40° C. in 40 minutes. Afterwards scalding agitationcontinued for further 20 minutes. Then the whey was drained off and thecurd was further drained until pH of 5.2-5.3 was reached. The curd wasmilled, salted and filled into moulds. The cheeses were pressed in moldsfor 17 hours. One cheese of about 14 kg was obtained from each vat of150 kg vat milk.

After pressing the cheeses were removed from the moulds and vacuumpacked in Cryovac® BL1L plastic bags (Cryovac, St. Neots, Belgium) andstored at 9° C. for a defined storage time.

The evolution of pH values was followed during cheese make as shownbelow:

Time [hours] Control Cheese with Cheese with after cheese adjunctadjunct culture Step in culture without cultre mutant mother strainmanufacture addition adjunct DSM19501 DSM19500 Add milk 0:00 6.62 6.626.62 Whey off 2:45 6.39 6.35 6.31 2. turn 3:20 5.85 5.79 5.74 3. turn3:40 5.59 5.54 5.45 4. turn 4:05 5.39 5.36 5.24* 5. turn 4:15 5.34 5.27*6. turn 4:30 5.29* 1 week old cheese 5.26 5.26 5.08 *milling pH reached

Milling pH was reached 25 minutes earlier when the Lactobacillushelveticus mother strain DSM19500 was used as adjunct in comparison tothe reference cheese without use of Lactobacillus helveticus adjunctculture.

The use of the non-acidifying mutant DSM19501 had only minor influenceon milk acidification, see FIG. 3.

The pH at 1 week age was significantly lower in the cheese made with theLactobacillus helveticus mother strain DSM19500 as adjunct.

Sensory Evaluation

An expert panel (5 panelists) evaluated the cheeses organolepticallyafter 10 weeks of ripening. The trays with cheese samples were temperedin a thermostatic upboard at 12° C. before the sensory evaluation. Thepanelists were asked to rate each cheese on a 15 cm undifferentiatedscale for each sensory attribute (0 being low intensity and 15 beinghigh intensity). The control cheese made without adjunct culture was thereference cheese for sensory profiling. A consensus sensory profile ofthe reference cheese was established first.

Than the two cheeses containing adjunct cultures and the control cheesewithout adjunct culture were evaluated. A randomized three-digitidentification code was given to each of the samples. Consensus profileof each cheese was established in comparison to the reference. Thevalues given for the control cheese in the table below are averages ofthe first profiling as reference cheese and the second evaluation amongthe coded samples.

Flavour profiles of Cheddar cheeses at 10 weeks age (scale 0-15 cm)

Control cheese Cheese with Cheese with without adjunct adjunct cultureadjunct culture culture DSM19501 DSM19500 Boiled milk 6.50 6.00 4.00Whey 4.00 4.00 4.00 Milk fat 9.13 4.00 6.50 Sulfur 2.75 2.75 1.50 Brothy5.50 5.25 7.75 Farmhouse 4.00 4.25 5.25 Sour 6.50 6.50 6.50 Bitter 5.254.00 3.50 Salt 5.63 6.50 7.75 Sweet 4.63 6.50 7.50

The flavour profiles of the three cheeses (except descriptors “whey” and“sour” having the same values for all three cheeses) are shown in FIG.4.

The control cheese made without adjunct culture was related to thesensory descriptors “boiled milk”, “bitter” and “milk fat”. In contrast,the two cheeses made with adjunct Lactobacillus helveticus strains weredifferent from the control cheese. Sensory descriptors like “salty” and“sweet” dominated more and those cheeses were described as significantlyless bitter than the control cheese.

It was thereby demonstrated that non-acidifying Lactobacillus helveticusstrains can be successfully used as adjunct cultures to improve flavourand/or texture and/or taste in cheese.

1-23. (canceled)
 24. A process for improving the texture and/or tasteand/or flavour of cheese, comprising: adding to milk a lactic acidbacteria culture comprising a strain belonging to a genus selected fromthe group consisting of: Lactococcus, Leuconostoc, Pediococcus,Streptococcus, and Enterococcus, and a non-acidifying Lactobacillushelveticus strain, wherein the non-acidifying Lactobacillus helveticusstrain is not able to lower the pH more than 1.5 pH Units from start pH6.5 after 10 hours incubation at 37° C. when inoculated from a freshover night culture at inoculation dose 1.5×10⁷ cfu/ml into 200 ml ofmilk, and a coagulant, such as a milk-clotting enzyme; and then heatingthe resultant mixture to a temperature in the range of 30 to 45 degreesC.
 25. The process of claim 24, which comprises, after heating themixture to a temperature in the range of 30 to 45 degrees C., holdingthe mixture at that temperature range for 5 to 70 minutes immediatelybefore whey removal or pre-pressing under whey.
 26. The process of claim24, wherein the non-acidifying Lactobacillus helveticus strain is notable to lower the pH more than 1.5 pH Units from start pH 6.5 after 10hours incubation at 37° C. when inoculated from a fresh over nightculture at inoculation dose 1.5×10⁷ cfu/ml into 200 of ml milk preparedfrom 9.5% skim milk powder rehydrated in water (heat treated at 140°C./8 sec and 100° C./30 min).
 27. The process of claim 26, wherein thenon-acidifying Lactobacillus helveticus strain is not able to lower thepH more than 1.3 pH Units from start pH 6.5 after 10 hours incubation at37° C. when inoculated from a fresh over night culture at inoculationdose 1.5×10⁷ cfu/ml into 200 ml of milk.
 28. The process of claim 24,wherein the non-acidifying Lactobacillus helveticus strain is a mutantof an acidifying strain, the mutant having an at least as high cell wallbound protease activity as the mother strain.
 29. The process of claim24, wherein the non-acidifying Lactobacillus helveticus strain is amutant of an acidifying Lactobacillus helveticus strain.
 30. The processof claim 29, wherein the non-acidifying Lactobacillus helveticus strainis the mutant DSM
 19501. 31. The process according to claim 24, whereinthe culture comprises a strain selected from the group consisting ofLactococcus lactis, Leuconostoc mesenteroides, Pediococcus pentosaceus,Lactobacillus casei, Lactobacillus paracasei, Streptococcusthermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus delbrueckii subsp. lactis and Lactobacillusacidophilus.
 32. The process of claim 24, wherein the cheese to be madeis a cheddar type cheese or a continental type cheese, including low-fatcheese.
 33. A cheese obtainable by the process of claim 24, such as acheddar type cheese.
 34. A Lactobacillus helveticus strain selected fromthe group of DSM 19500, DSM 19501, DSM 18879, DSM 18880, DSM 18881, DSM18871, DSM 18872, DSM 18873, DSM 18883, DSM 18884, and a mutant of anyof the foregoing that is not able to lower the pH more than 1.5 pH Unitsfrom start pH 6.5 after 10 hours incubation at 37° C. when inoculatedfrom a fresh over night culture at inoculation dose 1.5×10⁷ cfu/ml into200 ml of milk.
 35. A Lactobacillus helveticus strain of claim 34,wherein said strain is said mutant.
 36. The Lactobacillus helveticusstrain of claim 34, wherein said strain is selected from the group ofDSM 19501 and a mutant or variant thereof.